Volume 19, Issue 1, Pages 135-145 (January 2014) Epigenetic Regulation of the DLK1-MEG3 MicroRNA Cluster in Human Type 2 Diabetic Islets  Vasumathi Kameswaran, Nuria C. Bramswig, Lindsay B. McKenna, Melinda Penn, Jonathan Schug, Nicholas J. Hand, Ying Chen, Inchan Choi, Anastassios Vourekas, Kyoung-Jae Won, Chengyang Liu, Kumar Vivek, Ali Naji, Joshua R. Friedman, Klaus H. Kaestner  Cell Metabolism  Volume 19, Issue 1, Pages 135-145 (January 2014) DOI: 10.1016/j.cmet.2013.11.016 Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 The Imprinted Chromosome 14q32 miRNA Cluster Is Downregulated in T2DM Islets (A and B) Expression levels of the 15 most abundant miRNAs in (A) three nondiabetic and (B) four T2DM human islets as identified by sRNA sequencing. (C) Differentially expressed miRNAs between non-T2DM (n = 3) and T2DM (n = 4) islets, identified by a FDR of 20% and minimum fold change of 1.5×. miRNAs upregulated and downregulated in T2DM islets are highlighted. (D) Distribution of expression levels of miRNAs identified as significantly differentially expressed by sRNA sequencing across clustered samples. (E) Relative expression of miRNAs in the Chr 14q32 cluster as determined by Taqman qPCR of 14 non-T2DM and 10 T2DM human islets. p value was calculated using two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005. Error bars indicate SEM. See also Figure S1 and Table S2. Cell Metabolism 2014 19, 135-145DOI: (10.1016/j.cmet.2013.11.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Chr 14q32 miRNAs Are Highly and Specifically Expressed in Human β Cells (A) Average expression of Chr 14q32 cluster miRNAs in human α and β cells. (B) Expression of Chr 14q32 miRNAs (minimal expression 50 reads per million [RPM]) in sorted human α and β cells. (C) Genome browser image of histone modification marks H3K4me3 (n = 4) and H3K27me3 (n = 3) at the MEG3 promoter (chromosomal location marked on top) of sorted α and β cells from healthy human donors. Gray bars represent sequence conservation. Positions of the miRNA cluster and other nearby transcripts are shown. See also Figure S2 and Table S3. Cell Metabolism 2014 19, 135-145DOI: (10.1016/j.cmet.2013.11.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Increased Methylation of the MEG3 Differentially Methylated Region in T2DM Islets (A) Schematic representation of DLK1-MEG3 locus differentially methylated regions (DMRs) with allele-specific gene expression depicted. Regions analyzed for Figures 3C and 3D are marked as green bars. (B) Methylation-specific PCR for MEG3 promoter methylation in two T2DM and two nondiabetic donors’ islets. The methylated band (Me) is 160 bp and the unmethylated band (Un) is 120 bp. (C and D) Percent methylation was determined for multiple CpGs in the (C) IG-DMR and (D) MEG3-DMR in five nondiabetic and nine T2DM donors’ islets by pyrosequencing of bisulfite-converted DNA. Each bar represents an individual CpG, and the regions refer back to schema in part (A). p value calculated using Student’s t test. ∗∗p < 0.005 and ∗∗∗p < 0.001. Error bars indicate SEM. See also Figure S3. Cell Metabolism 2014 19, 135-145DOI: (10.1016/j.cmet.2013.11.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Identification of miRNA Targets in Human Islets by HITS-CLIP (A) Schema of HITS-CLIP procedure and chimeric reads ligation. (B) Average read coverage of all HITS-CLIP target mRNA fragments over a standardized mRNA. (C) Argonaute footprint distribution across target library mRNAs in human islets. (D) Targeting of human IAPP mRNA by miR-432 and miR-376a was validated by luciferase reporter assays. Vectors with or without the 3′ UTR of IAPP were cotransfected with either empty pCAG-eGFP vector or miR-432 and miR-376a. Error bars indicate ±SEM. ∗∗∗p value calculated using Student’s t test. p = 1.8 × 10−5. See Tables S4 and S5. Cell Metabolism 2014 19, 135-145DOI: (10.1016/j.cmet.2013.11.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Determination of Direct miRNA:mRNA Targeting Relationship from Chimeric Reads Deep sequencing of our Argonaute HITS-CLIP library identified thousands of chimeric reads, consisting of a mature miRNA and a target mRNA fragment. (A) The 15 most abundant mRNAs found in chimeric reads in human islets. (B) The 15 most highly miRNA-regulated mRNAs in chimeric reads. The regulatory load ratio is the relative Ago-associated mRNA fraction of the chimeric reads, defined as the ratio of their sequence counts to their normalized abundance in human islets. (C) The 15 most abundant miRNAs found in chimeric reads in human islets. (D) Significantly enriched gene ontology biological processes in targets of human islet miRNAs. (E) Pie chart representation of distribution of mRNA regions found in chimeras with miRNAs. (F) Average read coverage of chimeric mRNA fragments across an mRNA divided into 150 equal bins. See Figure S4 and Table S6. Cell Metabolism 2014 19, 135-145DOI: (10.1016/j.cmet.2013.11.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Validation of the miR-495:TP53INP1 Targeting Relationship A β cell apoptotic factor, TP53INP1, is regulated by miR-495. (A) The sequence of the miR-495 (orange) and TP53INP1 3′ UTR (green) chimera. Folded confirmation with base pairing between the miRNA and 3′ UTR is indicated below. (B) Relative levels of TP53INP1 mRNA between T2DM and non-T2DM islet samples. Error bars indicate ±SEM. (C) Anticorrelation between normalized TP53INP1 and miR-495 in nine T2DM islet donor samples. (D) Targeting of human TP53INP1 mRNA by miR-495 was validated by luciferase reporter assays. Vectors with or without the 3′ UTR of TP53INP1 were cotransfected with either scramble or miR-495 mimics. Error bars indicate ±SEM. p value calculated using Student’s t test. ∗∗∗p = 1.94 × 10−5. (E) Relative mRNA levels of TP53INP1 and ONECUT1 (normalized to the average of HPRT and β-actin transcript levels) in human islets transduced with lentivirus encoding tough decoy constructs for either scramble sequence (TuDctrl) or miRNA-495 (TuD495). Error bars indicate ±SEM. ∗∗p = 0.0076, n = 3. Cell Metabolism 2014 19, 135-145DOI: (10.1016/j.cmet.2013.11.016) Copyright © 2014 Elsevier Inc. Terms and Conditions